- Email: [email protected]
Nitric oxide in plants
Phytochemistry 54 (2000) 1±4
www.elsevier.com/locate/phytochem
Molecules of Interest
Nitric oxide in plants To NO or not to NO Przemysl/ aw Wojtaszek* Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, PoznanÂ, Poland Received 10 January 2000; accepted 10 January 2000
Abstract The current knowledge on the occurrence and activity of NO in plants is reviewed. The multiplicity of nitrogen monoxide species and implications for dierentiated reactivity are indicated. Possible sources of NO are evaluated, and the evidence for the presence of nitric oxide synthase in plants is summarised. The regulatory role of NO in plant development and in plant interactions with microorganisms, involving an interplay with other molecules, like ethylene or reactive oxygen species is demonstrated. Finally, some other suggestions on potential functions of NO in plants are indicated. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Nitrogen monoxide; Nitric oxide; Reactivity; Biosynthesis; Nitric oxide synthase; Biological function
1. Introduction For more than 50 years, the only gaseous signalling molecule in the living world known to science was the plant hormone, ethylene. The 1998 Nobel Prize for Medicine heralded the establishment of another, even a smaller one, player of this kind in mammalian cells Ð nitric oxide. This relatively stable free radical has been initially identi®ed as an endothelium-derived relaxation factor, and later implicated in signal transduction pathways controlling neurotransmission, cell proliferation, platelet inhibition, programmed cell death, and host responses to infection (Wink and Mitchell, 1998). The presence of nitrogen monoxide in plants has been known for some time (Leshem, 1996), and its involvement in regulation of plant growth (Gouvea et al., 1997) and phytoalexin accumulation (Noritake et al., 1996) has been demonstrated. It was however the appearance of two ground-breaking papers (Delledonne et al., 1998; Durner et al., 1998) * Tel.: +48-61-852-85-03; fax: +48-61-852-05-32. E-mail address: [email protected] (P. Wojtaszek).
that demonstrated clearly its regulatory role in plant biology, in this case during interactions with pathogenic microorganisms. Recent excellent reviews of this topic (Bolwell, 1999; Durner and Klessig, 1999), allow me to consider only selected important points of ``chemical biology of nitric oxide'' (Wink and Mitchell, 1998) in plants. 2. Multiplicity of nitrogen monoxide chemistry Current views ascribe most of physiological eects of NO action to its radical species Ð nitric oxide. Based on analogies with animal systems, the same assumption is becoming prevalent in studies of plants, although direct detection and determination of NO forms present in planta are very scarce (e.g. Mathieu et al., 1998). It should be noted however that the NO chemistry is much more complicated and involves an interplay between three species diering in physical properties and chemical reactivity: nitrosonium cation (NO+), nitric oxide radical (NO ), and nitroxyl anion (NOÿ). Their chemistry is not recognised to that extent as the chemistry and implications of that chemistry for
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 1 - 9 4 2 2 ( 0 0 ) 0 0 0 5 6 - X
2
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
biological activities of respective dioxygen counterparts: O2, superoxide radical anion ( Oÿ 2 ), and hydrogen peroxide (H2O2) (Stamler et al., 1992). There are also important consequences for methodological design of experiments aimed at elucidation of the role of NO in plants, and a cautionary note seems appropriate in this place. Some conclusions are drawn on the basis of experimental results obtained with NOreleasing compounds, and the implicit assumption is that the NO species released is nitric oxide (e.g. Delledonne et al., 1998). Although compounds with clearly de®ned ability to release NO are now available, some other commonly used complexes, like nitroprusside are in fact iron±nitrosyl substances with strong NO+ character (cited after Stamler et al., 1992). 3. Origin of nitrogen monoxide in plants There are many possible sources of nitrogen monoxide (Fig. 1). Although in animals NO is generated almost exclusively by nitric oxide synthase (NOS, EC 1.14.13.39), in bacteria, fungi, and plants the presence of NO, like the presence of ROS, is intimately implicated in their metabolism, and in fact is one of the elements of nitrogen cycling on Earth. Nitri®cation/ denitri®cation cycles provide NO as a by-product of N2O oxidation into the atmosphere. Plants not only react to the atmospheric or soil NO, but are also able to emit substantial amounts of NO (see Durner and Klessig, 1999). Thus, NO could be generated by nonenzymatic mechanisms, e.g. via chemical reduction of NOÿ 2 at acidic pH or by carotenoids in the presence of
light (Cooney et al., 1994). The major origin of NO production in plants however is probably through the action of NAD(P)H-dependent nitrate or nitrite reductases (Yamasaki et al., 1999). In mammals, NOS catalyses a ®ve-electron oxidation of a terminal guanidino nitrogen of arginine, and insertion of a pair of oxygen atoms (from two activated O2 molecules) yields citrulline and NO. The reaction is supported by NADPH and ¯avins, and thiol and tetrahydrobiopterin are additional cofactors. Mechanistically, the enzyme is similar to cytochrome P450. The existence of NOS in plants was inferred from immunoreactivity of protein extracts (Sen and Cheema, 1995), and indications of NOS-type activity in plant tissues (Cueto et al., 1996; Ninnemann and Maier, 1996). Subsequently, NOS induction during plant±pathogen interactions (Delledonne et al., 1998; Durner et al., 1998) and growth phase-dependent NOS localisation (Ribeiro et al., 1999) have been demonstrated. However, the identity of plant enzyme with mammalian-type NOS is far from proved. Both the protein, and the gene(s) coding for the enzyme have not been identi®ed in plants yet. Moreover, as Durner and Klessig (1999) rightly pointed out, there are no data whether tetrahydrobiopterin is synthesised by plants. Con¯icting results have also been obtained with regard to Ca2+-dependence. While NOS activity in lupin roots was Ca2+-dependent, similarly to constitutive NOS isoforms in mammals, NOS activity in lupin nodules was Ca2+-independent thus resembling mammalian inducible isoforms (Cueto et al., 1996). On the other hand, an enzyme induced in plants by pathogenic infection was Ca2+-dependent (Delledonne et al.,
Fig. 1. Possible sources of nitrogen monoxide. NO is generated by the action of nitric oxide synthase (NOS). Other major origins of NO are the reactions utilising NOÿ 2 : non-enzymatic reductions either at acidic pH or light-driven in the presence of carotenoids, and enzymatic catalysed by NAD(P)H-dependent nitrate (NR) or nitrite (NiR) reductases. It could also be a by-product of denitri®cation, nitrate assimilation and/or respiration. Nitri®cation of NH+ 4 is the major source of N2O emitted to the atmosphere where it might be further oxidised to NO and NO2.
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
1998). Dierentiated sensitivity of plant and mammalian enzymes to NOS inhibitors (Durner et al., 1998) made the analyses of plant enzyme even more complicated. Moreover, the existence of multiple calmodulin isoforms aecting dierently NOS activity opens the possibility of more complex regulatory circuits and variable branching of signalling pathways (Cho et al., 1998). 4. Role of nitrogen monoxide in plants 4.1. Molecular targets Due to dierentiated chemical reactivity of NO species, there are many potential targets of NO action. From the biological point of view, the best characterised are the reactions of NO and NO+. Nitric oxide ÿ reacts with Oÿ 2 yielding peroxynitrite (ONOO ), the major toxic reactive nitrogen oxide species (Stamler et al., 1992). As the apoplastic route for NO transport has been proposed (Leshem, 1996), and superoxide dismutase (SOD) is a major regulator of Oÿ 2 levels, recent demonstration of exocellular SOD isoforms in plants (Schinkel et al., 1998) suggested the existence of an exocellular system preventing the formation of ONOOÿ. Similarly, mitochondrial alternative oxidase was implicated in restriction of Oÿ 2 generation under conditions of cytochrome c oxidase inhibition by NO again limiting oxidative damage of the cells (Millar and Day, 1997). Other potential targets of NO action include proteins containing transition metal ions either as haem or non-haem complexes, mainly iron±sulphur centres in the latter case. NO+ is involved in addition and substitution reactions with nucleophiles, and most often nitrosations occur at ÿS, ÿN, ÿO, and ÿC centres of organic molecules (Stamler et al., 1992). This multiplicity of potential reactions has dual consequences. Firstly, depending on the location, structure, and reactivity of the product formed, it could be regarded either as an end result of NO degradation or as an element of NO-transporting/storage pool. Secondly, especially due to reactions with other free radicals, NO could be treated as a molecule breaking the propagation of radical-mediated oxidation chains thus limiting cellular damage (Beligni and Lamattina, 1999). 4.2. Regulatory interplay Despite the multiplicity of potential targets, only few proteins have as yet been demonstrated to be regulated by interactions with NO. The most interesting candidate is guanylate cyclase and the resulting activation of cGMP-dependent signalling pathway. This relation was demonstrated in TMV-infected tobacco and an
3
involvement of another NO-dependent signalling molecule: cyclic ADP-ribose was also detected (Durner et al., 1998). As NO easily forms iron±nitrosyl complexes with haem iron, it is not surprising that NO eect on the functioning of plant peroxidases participating in wall ligni®cation has been recently revealed (Ferrer and Ros Barcelo, 1999). The most interesting observations are those on the involvement of NO in regulation of plant defence responses to pathogen infection (Noritake et al., 1996; Delledonne et al., 1998; Durner et al., 1998). There seems to exist a regulatory interplay between NO and ROS, and salicylic acid is also playing its role. Direct reactions of NO and ROS might be involved as well as indirect through activation/repression of respective signalling pathways leading to changes at the gene expression and protein activity levels (reviewed by Bolwell, 1999; Durner and Klessig, 1999). However, as we are at the beginning of the road in this ``web of signals'' there are many more questions to be answered and care should be taken to avoid misinterpretations. It is thus worth reminding that the biochemistry of reactive NO and oxygen species might be intimately intertwined as e.g. under conditions of arginine depletion, NOS is synthesising H2O2 (Stamler et al., 1992). On the other hand, diphenylene iodonium Ð an inhibitor considered speci®c for the ROS-generating NADPH oxidase is also an inhibitor of H2O2-generating activity of peroxidases and moreover it inhibits NOS activity (reviewed by Bolwell and Wojtaszek, 1997; Bolwell, 1999). Similarly, some inhibitors of guanylate cyclase Ð a major NO target, acting through oxidation of thiols and transition metals, are even more potent NOS inhibitors (Hausladen and Stamler, 1998). Finally, on the basis of many pharmacological data, and in continuation of previous suggestions (Wojtaszek, 1997), the role of phenolic compounds as regulators of the extent of NO action should also be not overlooked. 4.3. Potential functions Relatively little is known about the biological functions of NO in plants. Some of them might be inferred from the data gathered on mammalian cells, but, mainly due to dierences in developmental processes, some of them would be probably unique to plants. Thus, like in mammalian cells there are clear indications that NO is involved in signalling events during interactions with microorganisms, either symbiotic (Cueto et al., 1996; Mathieu et al., 1998) or pathogenic (Noritake et al., 1996; Delledonne et al., 1998; Durner et al., 1998). On the other hand, NO is probably a regulatory molecule during plant development aecting growth (Gouvea et al., 1997), and activity of enzymes involved in cell dierentiation (Ferrer and Ros Bar-
4
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
celo, 1999). Endogenous NO is even sometimes considered as a plant growth regulator (Leshem, 1996), and the evidence for an interplay between NO and ethylene Ð a hormonal system unique to plants Ð in the regulation of maturation and senescence of plant tissues has been already evidenced (Leshem et al., 1998). The ®eld of chemical biology of nitric oxide in plants is rapidly expanding, and it is hoped that the near future will bring some more answers. Even now a quick survey through the literature reveals other potential analogies with mammalian systems oering a promising prospectus for future studies in plants, and two examples will be mentioned here. Plant oligosaccharides are able to stimulate the release of NO from murine eector cells (Hirazumi and Furusawa, 1999) suggesting potential interplay between NO and cell wall-derived oligosaccharins. The identi®cation of plant homologues of NRAMP1 protein provides an indication that plants might be equipped with a system similar to the animal one where NRAMP1 has been identi®ed as a controller of innate immunity and is speculated to function as a nitrite transporter supplying substrate for NO synthesis (reviewed by Nathan, 1995). Acknowledgements The funding by State Committee for Scienti®c Research (grant 6 P04C 022 13) is gratefully acknowledged. References Beligni, M.V., Lamattina, L., 1999. Is nitric oxide toxic or protective? Trends in Plant Science 4, 299±300. Bolwell, G.P., 1999. Role of reactive oxygen species and NO in plant defence responses. Current Opinion in Plant Biology 2, 287±294. Bolwell, G.P., Wojtaszek, P., 1997. Mechanisms for the generation of reactive oxygen species in plant defence Ð a broad perspective. Physiological and Molecular Plant Pathology 51, 347±366. Cho, M.J., Vaghy, P.L., Kondo, R., Lee, S.H., Davis, J.P., Rehl, R., Heo, W.D., Johnson, J.D., 1998. Reciprocal regulation of mammalian nitric oxide synthase and calcineurin by plant calmodulin isoforms. Biochemistry 37, 15,593±15,597. Cooney, R.V., Harwood, P.J., Custer, L.J., Franke, A.A., 1994. Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids. Environmental Health Perspectives 102, 460±462. Cueto, M., Hernandez-Perera, O., Martin, R., Bentura, M.L., Rodrigo, J., Lamas, S., Golvano, M.P., 1996. Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus. FEBS Letters 398, 159±164. Delledonne, M., Xia, Y.J., Dixon, R.A., Lamb, C., 1998. Nitric
oxide functions as a signal in plant disease resistance. Nature 394, 585±588. Durner, J., Klessig, D.F., 1999. Nitric oxide as a signal in plants. Current Opinion in Plant Biology 2, 369±374. Durner, J., Wendehenne, D., Klessig, D.F., 1998. Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADPribose. Proceedings of the National Academy of Sciences USA 95, 10,328±10,333. Ferrer, M.A., Ros Barcelo, A., 1999. Dierential eects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans. Plant, Cell and Environment 22, 891±897. Gouvea, C.M.C.P., Souza, J.F., Magalhaes, A.C.N., Martins, I.S., 1997. NO-releasing substances that induce growth elongation in maize root segments. Plant Growth Regulation 21, 183±187. Hausladen, A., Stamler, J.S., 1998. Nitric oxide in plant immunity. Proceedings of the National Academy of Sciences USA 95, 10,345±10,347. Hirazumi, A., Furusawa, E., 1999. An immunomodulatory polysaccharide-rich substance from the fruit juice of Morinda citri¯ora (Noni) with antitumour activity. Phytotherapy Research 13, 380± 388. Leshem, Y.Y., 1996. Nitric oxide in biological systems. Plant Growth Regulation 18, 155±169. Leshem, Y.Y., Wills, R.B.H., Ku, V.V.V., 1998. Evidence for the function of the free radical gas Ð nitric oxide (NO ) Ð as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiology and Biochemistry 36, 825±833. Mathieu, C., Moreau, S., Frendo, P., Puppo, A., Davies, M.J., 1998. Free Radical Biology and Medicine 24, 1242±1249. Millar, A.H., Day, D.A., 1997. Alternative solutions to radical problems. Trends in Plant Science 2, 289±290. Nathan, C., 1995. Natural resistance and nitric oxide. Cell 82, 873± 876. Ninnemann, H., Maier, J., 1996. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochemistry and Photobiology 64, 393±398. Noritake, T., Kawakita, K., Doke, N., 1996. Plant & Cell Physiology 37, 113±116. Ribeiro, E.A., Cunha, F.Q., Tamashiro, W.M.S.C., Martins, I.S., 1999. Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells. FEBS Letters 445, 283±286. Schinkel, H., Streller, S., Wingsle, G., 1998. Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine. Journal of Experimental Botany 49, 931±936. Sen, S., Cheema, I.R., 1995. Nitric oxide synthase and calmodulin immunoreactivity in plant embryonic tissue. Biochemical Archives 11, 221±227. Stamler, J.S., Singel, D.J., Loscalzo, J., 1992. Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898±1902. Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology and Medicine 25, 434± 456. Wojtaszek, P., 1997. Oxidative burst: an early plant response to pathogen infection. Biochemical Journal 322, 681±692. Yamasaki, H., Sakihama, Y., Takahashi, S., 1999. An alternative pathway for nitric oxide production in plants: new features of an old enzyme. Trends in Plant Science 2, 128±129.
www.elsevier.com/locate/phytochem
Molecules of Interest
Nitric oxide in plants To NO or not to NO Przemysl/ aw Wojtaszek* Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, PoznanÂ, Poland Received 10 January 2000; accepted 10 January 2000
Abstract The current knowledge on the occurrence and activity of NO in plants is reviewed. The multiplicity of nitrogen monoxide species and implications for dierentiated reactivity are indicated. Possible sources of NO are evaluated, and the evidence for the presence of nitric oxide synthase in plants is summarised. The regulatory role of NO in plant development and in plant interactions with microorganisms, involving an interplay with other molecules, like ethylene or reactive oxygen species is demonstrated. Finally, some other suggestions on potential functions of NO in plants are indicated. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Nitrogen monoxide; Nitric oxide; Reactivity; Biosynthesis; Nitric oxide synthase; Biological function
1. Introduction For more than 50 years, the only gaseous signalling molecule in the living world known to science was the plant hormone, ethylene. The 1998 Nobel Prize for Medicine heralded the establishment of another, even a smaller one, player of this kind in mammalian cells Ð nitric oxide. This relatively stable free radical has been initially identi®ed as an endothelium-derived relaxation factor, and later implicated in signal transduction pathways controlling neurotransmission, cell proliferation, platelet inhibition, programmed cell death, and host responses to infection (Wink and Mitchell, 1998). The presence of nitrogen monoxide in plants has been known for some time (Leshem, 1996), and its involvement in regulation of plant growth (Gouvea et al., 1997) and phytoalexin accumulation (Noritake et al., 1996) has been demonstrated. It was however the appearance of two ground-breaking papers (Delledonne et al., 1998; Durner et al., 1998) * Tel.: +48-61-852-85-03; fax: +48-61-852-05-32. E-mail address: [email protected] (P. Wojtaszek).
that demonstrated clearly its regulatory role in plant biology, in this case during interactions with pathogenic microorganisms. Recent excellent reviews of this topic (Bolwell, 1999; Durner and Klessig, 1999), allow me to consider only selected important points of ``chemical biology of nitric oxide'' (Wink and Mitchell, 1998) in plants. 2. Multiplicity of nitrogen monoxide chemistry Current views ascribe most of physiological eects of NO action to its radical species Ð nitric oxide. Based on analogies with animal systems, the same assumption is becoming prevalent in studies of plants, although direct detection and determination of NO forms present in planta are very scarce (e.g. Mathieu et al., 1998). It should be noted however that the NO chemistry is much more complicated and involves an interplay between three species diering in physical properties and chemical reactivity: nitrosonium cation (NO+), nitric oxide radical (NO ), and nitroxyl anion (NOÿ). Their chemistry is not recognised to that extent as the chemistry and implications of that chemistry for
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 1 - 9 4 2 2 ( 0 0 ) 0 0 0 5 6 - X
2
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
biological activities of respective dioxygen counterparts: O2, superoxide radical anion ( Oÿ 2 ), and hydrogen peroxide (H2O2) (Stamler et al., 1992). There are also important consequences for methodological design of experiments aimed at elucidation of the role of NO in plants, and a cautionary note seems appropriate in this place. Some conclusions are drawn on the basis of experimental results obtained with NOreleasing compounds, and the implicit assumption is that the NO species released is nitric oxide (e.g. Delledonne et al., 1998). Although compounds with clearly de®ned ability to release NO are now available, some other commonly used complexes, like nitroprusside are in fact iron±nitrosyl substances with strong NO+ character (cited after Stamler et al., 1992). 3. Origin of nitrogen monoxide in plants There are many possible sources of nitrogen monoxide (Fig. 1). Although in animals NO is generated almost exclusively by nitric oxide synthase (NOS, EC 1.14.13.39), in bacteria, fungi, and plants the presence of NO, like the presence of ROS, is intimately implicated in their metabolism, and in fact is one of the elements of nitrogen cycling on Earth. Nitri®cation/ denitri®cation cycles provide NO as a by-product of N2O oxidation into the atmosphere. Plants not only react to the atmospheric or soil NO, but are also able to emit substantial amounts of NO (see Durner and Klessig, 1999). Thus, NO could be generated by nonenzymatic mechanisms, e.g. via chemical reduction of NOÿ 2 at acidic pH or by carotenoids in the presence of
light (Cooney et al., 1994). The major origin of NO production in plants however is probably through the action of NAD(P)H-dependent nitrate or nitrite reductases (Yamasaki et al., 1999). In mammals, NOS catalyses a ®ve-electron oxidation of a terminal guanidino nitrogen of arginine, and insertion of a pair of oxygen atoms (from two activated O2 molecules) yields citrulline and NO. The reaction is supported by NADPH and ¯avins, and thiol and tetrahydrobiopterin are additional cofactors. Mechanistically, the enzyme is similar to cytochrome P450. The existence of NOS in plants was inferred from immunoreactivity of protein extracts (Sen and Cheema, 1995), and indications of NOS-type activity in plant tissues (Cueto et al., 1996; Ninnemann and Maier, 1996). Subsequently, NOS induction during plant±pathogen interactions (Delledonne et al., 1998; Durner et al., 1998) and growth phase-dependent NOS localisation (Ribeiro et al., 1999) have been demonstrated. However, the identity of plant enzyme with mammalian-type NOS is far from proved. Both the protein, and the gene(s) coding for the enzyme have not been identi®ed in plants yet. Moreover, as Durner and Klessig (1999) rightly pointed out, there are no data whether tetrahydrobiopterin is synthesised by plants. Con¯icting results have also been obtained with regard to Ca2+-dependence. While NOS activity in lupin roots was Ca2+-dependent, similarly to constitutive NOS isoforms in mammals, NOS activity in lupin nodules was Ca2+-independent thus resembling mammalian inducible isoforms (Cueto et al., 1996). On the other hand, an enzyme induced in plants by pathogenic infection was Ca2+-dependent (Delledonne et al.,
Fig. 1. Possible sources of nitrogen monoxide. NO is generated by the action of nitric oxide synthase (NOS). Other major origins of NO are the reactions utilising NOÿ 2 : non-enzymatic reductions either at acidic pH or light-driven in the presence of carotenoids, and enzymatic catalysed by NAD(P)H-dependent nitrate (NR) or nitrite (NiR) reductases. It could also be a by-product of denitri®cation, nitrate assimilation and/or respiration. Nitri®cation of NH+ 4 is the major source of N2O emitted to the atmosphere where it might be further oxidised to NO and NO2.
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
1998). Dierentiated sensitivity of plant and mammalian enzymes to NOS inhibitors (Durner et al., 1998) made the analyses of plant enzyme even more complicated. Moreover, the existence of multiple calmodulin isoforms aecting dierently NOS activity opens the possibility of more complex regulatory circuits and variable branching of signalling pathways (Cho et al., 1998). 4. Role of nitrogen monoxide in plants 4.1. Molecular targets Due to dierentiated chemical reactivity of NO species, there are many potential targets of NO action. From the biological point of view, the best characterised are the reactions of NO and NO+. Nitric oxide ÿ reacts with Oÿ 2 yielding peroxynitrite (ONOO ), the major toxic reactive nitrogen oxide species (Stamler et al., 1992). As the apoplastic route for NO transport has been proposed (Leshem, 1996), and superoxide dismutase (SOD) is a major regulator of Oÿ 2 levels, recent demonstration of exocellular SOD isoforms in plants (Schinkel et al., 1998) suggested the existence of an exocellular system preventing the formation of ONOOÿ. Similarly, mitochondrial alternative oxidase was implicated in restriction of Oÿ 2 generation under conditions of cytochrome c oxidase inhibition by NO again limiting oxidative damage of the cells (Millar and Day, 1997). Other potential targets of NO action include proteins containing transition metal ions either as haem or non-haem complexes, mainly iron±sulphur centres in the latter case. NO+ is involved in addition and substitution reactions with nucleophiles, and most often nitrosations occur at ÿS, ÿN, ÿO, and ÿC centres of organic molecules (Stamler et al., 1992). This multiplicity of potential reactions has dual consequences. Firstly, depending on the location, structure, and reactivity of the product formed, it could be regarded either as an end result of NO degradation or as an element of NO-transporting/storage pool. Secondly, especially due to reactions with other free radicals, NO could be treated as a molecule breaking the propagation of radical-mediated oxidation chains thus limiting cellular damage (Beligni and Lamattina, 1999). 4.2. Regulatory interplay Despite the multiplicity of potential targets, only few proteins have as yet been demonstrated to be regulated by interactions with NO. The most interesting candidate is guanylate cyclase and the resulting activation of cGMP-dependent signalling pathway. This relation was demonstrated in TMV-infected tobacco and an
3
involvement of another NO-dependent signalling molecule: cyclic ADP-ribose was also detected (Durner et al., 1998). As NO easily forms iron±nitrosyl complexes with haem iron, it is not surprising that NO eect on the functioning of plant peroxidases participating in wall ligni®cation has been recently revealed (Ferrer and Ros Barcelo, 1999). The most interesting observations are those on the involvement of NO in regulation of plant defence responses to pathogen infection (Noritake et al., 1996; Delledonne et al., 1998; Durner et al., 1998). There seems to exist a regulatory interplay between NO and ROS, and salicylic acid is also playing its role. Direct reactions of NO and ROS might be involved as well as indirect through activation/repression of respective signalling pathways leading to changes at the gene expression and protein activity levels (reviewed by Bolwell, 1999; Durner and Klessig, 1999). However, as we are at the beginning of the road in this ``web of signals'' there are many more questions to be answered and care should be taken to avoid misinterpretations. It is thus worth reminding that the biochemistry of reactive NO and oxygen species might be intimately intertwined as e.g. under conditions of arginine depletion, NOS is synthesising H2O2 (Stamler et al., 1992). On the other hand, diphenylene iodonium Ð an inhibitor considered speci®c for the ROS-generating NADPH oxidase is also an inhibitor of H2O2-generating activity of peroxidases and moreover it inhibits NOS activity (reviewed by Bolwell and Wojtaszek, 1997; Bolwell, 1999). Similarly, some inhibitors of guanylate cyclase Ð a major NO target, acting through oxidation of thiols and transition metals, are even more potent NOS inhibitors (Hausladen and Stamler, 1998). Finally, on the basis of many pharmacological data, and in continuation of previous suggestions (Wojtaszek, 1997), the role of phenolic compounds as regulators of the extent of NO action should also be not overlooked. 4.3. Potential functions Relatively little is known about the biological functions of NO in plants. Some of them might be inferred from the data gathered on mammalian cells, but, mainly due to dierences in developmental processes, some of them would be probably unique to plants. Thus, like in mammalian cells there are clear indications that NO is involved in signalling events during interactions with microorganisms, either symbiotic (Cueto et al., 1996; Mathieu et al., 1998) or pathogenic (Noritake et al., 1996; Delledonne et al., 1998; Durner et al., 1998). On the other hand, NO is probably a regulatory molecule during plant development aecting growth (Gouvea et al., 1997), and activity of enzymes involved in cell dierentiation (Ferrer and Ros Bar-
4
P. Wojtaszek / Phytochemistry 54 (2000) 1±4
celo, 1999). Endogenous NO is even sometimes considered as a plant growth regulator (Leshem, 1996), and the evidence for an interplay between NO and ethylene Ð a hormonal system unique to plants Ð in the regulation of maturation and senescence of plant tissues has been already evidenced (Leshem et al., 1998). The ®eld of chemical biology of nitric oxide in plants is rapidly expanding, and it is hoped that the near future will bring some more answers. Even now a quick survey through the literature reveals other potential analogies with mammalian systems oering a promising prospectus for future studies in plants, and two examples will be mentioned here. Plant oligosaccharides are able to stimulate the release of NO from murine eector cells (Hirazumi and Furusawa, 1999) suggesting potential interplay between NO and cell wall-derived oligosaccharins. The identi®cation of plant homologues of NRAMP1 protein provides an indication that plants might be equipped with a system similar to the animal one where NRAMP1 has been identi®ed as a controller of innate immunity and is speculated to function as a nitrite transporter supplying substrate for NO synthesis (reviewed by Nathan, 1995). Acknowledgements The funding by State Committee for Scienti®c Research (grant 6 P04C 022 13) is gratefully acknowledged. References Beligni, M.V., Lamattina, L., 1999. Is nitric oxide toxic or protective? Trends in Plant Science 4, 299±300. Bolwell, G.P., 1999. Role of reactive oxygen species and NO in plant defence responses. Current Opinion in Plant Biology 2, 287±294. Bolwell, G.P., Wojtaszek, P., 1997. Mechanisms for the generation of reactive oxygen species in plant defence Ð a broad perspective. Physiological and Molecular Plant Pathology 51, 347±366. Cho, M.J., Vaghy, P.L., Kondo, R., Lee, S.H., Davis, J.P., Rehl, R., Heo, W.D., Johnson, J.D., 1998. Reciprocal regulation of mammalian nitric oxide synthase and calcineurin by plant calmodulin isoforms. Biochemistry 37, 15,593±15,597. Cooney, R.V., Harwood, P.J., Custer, L.J., Franke, A.A., 1994. Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids. Environmental Health Perspectives 102, 460±462. Cueto, M., Hernandez-Perera, O., Martin, R., Bentura, M.L., Rodrigo, J., Lamas, S., Golvano, M.P., 1996. Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus. FEBS Letters 398, 159±164. Delledonne, M., Xia, Y.J., Dixon, R.A., Lamb, C., 1998. Nitric
oxide functions as a signal in plant disease resistance. Nature 394, 585±588. Durner, J., Klessig, D.F., 1999. Nitric oxide as a signal in plants. Current Opinion in Plant Biology 2, 369±374. Durner, J., Wendehenne, D., Klessig, D.F., 1998. Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADPribose. Proceedings of the National Academy of Sciences USA 95, 10,328±10,333. Ferrer, M.A., Ros Barcelo, A., 1999. Dierential eects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans. Plant, Cell and Environment 22, 891±897. Gouvea, C.M.C.P., Souza, J.F., Magalhaes, A.C.N., Martins, I.S., 1997. NO-releasing substances that induce growth elongation in maize root segments. Plant Growth Regulation 21, 183±187. Hausladen, A., Stamler, J.S., 1998. Nitric oxide in plant immunity. Proceedings of the National Academy of Sciences USA 95, 10,345±10,347. Hirazumi, A., Furusawa, E., 1999. An immunomodulatory polysaccharide-rich substance from the fruit juice of Morinda citri¯ora (Noni) with antitumour activity. Phytotherapy Research 13, 380± 388. Leshem, Y.Y., 1996. Nitric oxide in biological systems. Plant Growth Regulation 18, 155±169. Leshem, Y.Y., Wills, R.B.H., Ku, V.V.V., 1998. Evidence for the function of the free radical gas Ð nitric oxide (NO ) Ð as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiology and Biochemistry 36, 825±833. Mathieu, C., Moreau, S., Frendo, P., Puppo, A., Davies, M.J., 1998. Free Radical Biology and Medicine 24, 1242±1249. Millar, A.H., Day, D.A., 1997. Alternative solutions to radical problems. Trends in Plant Science 2, 289±290. Nathan, C., 1995. Natural resistance and nitric oxide. Cell 82, 873± 876. Ninnemann, H., Maier, J., 1996. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochemistry and Photobiology 64, 393±398. Noritake, T., Kawakita, K., Doke, N., 1996. Plant & Cell Physiology 37, 113±116. Ribeiro, E.A., Cunha, F.Q., Tamashiro, W.M.S.C., Martins, I.S., 1999. Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells. FEBS Letters 445, 283±286. Schinkel, H., Streller, S., Wingsle, G., 1998. Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine. Journal of Experimental Botany 49, 931±936. Sen, S., Cheema, I.R., 1995. Nitric oxide synthase and calmodulin immunoreactivity in plant embryonic tissue. Biochemical Archives 11, 221±227. Stamler, J.S., Singel, D.J., Loscalzo, J., 1992. Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898±1902. Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology and Medicine 25, 434± 456. Wojtaszek, P., 1997. Oxidative burst: an early plant response to pathogen infection. Biochemical Journal 322, 681±692. Yamasaki, H., Sakihama, Y., Takahashi, S., 1999. An alternative pathway for nitric oxide production in plants: new features of an old enzyme. Trends in Plant Science 2, 128±129.